L-2-amino-4-methoxy-trans-3-butenoic acid

ABSTRACT

THE PRESENT INVENTION RELATES TO THE PREPARATION OF THE ANTIBACTERIAL SUBSTANCE L-2-AMINO-4-MEETHOXY - TRANS - 3BUTENOIC ACID BY CULTURING THE ORGANIXM PSEUDOMONAS AERUGINOSA.

United States Patent 3,739,022 L2-AMINO-4-METHOXY-TRANS- 3-BUTENOIC ACIDThomas Casimir Demny, Livingston, and James Parnell Scannell,Bloomfield, N.J., assignors to Hoifmann-La Roche Inc., Nutley, NJ. NoDrawing. Filed Oct. 20, 1971, Ser. No. 191,074 Int. Cl. C07c 101/04 US.Cl. 260--534 M 1 Claim ABSTRACT OF THE DISCLOSURE The present inventionrelates to the preparation of the antibacterial substanceL-2-amino-4-methoxy. trans 3- butenoic acid by culturing the organismPseudomonas aeruginosa.

The present invention relates to. a compound of the formula andpharmaceutically acceptable cationc saltsthereof.

The compound of the Formula I is prepared'by culturing a knownmicroorganism Pseudomonas aeruginosa ATCC 7700 in a fermentation mediumcontaining nutrient sources under submerged aerobic conditions, untilactivity against bacteria occurs and then isolating the soobtainedcompound of the Formula I from the fermentation broth. i

The conditions of fermentation are generally the same as conventionalmethods for producing a substance by fermentation. The fermentationmedium'contains the usual nutrient and mineral sources supplying carbon,nitrogen, and energy to the developing culture. Suitable nutrientsinclude, for example, carbohydrates such as, starch, dextrose, sucrose,glucose, molasses and nitrogen sources such as, soybean meal, yeast,meat extracts, corn steep liquor, distiller solubles, inorganic saltssuch as, calcium carbonate, ammonium sulfate, as well as trace elements.Suitably, the fermentation is permitted to proceed for from about 3 to10 days. p t

The compound of the Formula I can be isolated from the fermentationbroth in which it is prepared by adsorption into an anion exchangecolumn,'followed by elution with any suitable eluent preferablytrimethylammonium bicarbonate or any other equivalent volatile bufferedmedium. The isolationprocedure can beillustr'ated diagrammatically asfollows: i

Filtered broth Charcoal Filtrate II Evaporation Anion exchange Eluticnwith trimethyl ammonium bicarbonate ICC Eluate ConcentrationCrystallization from methanol Compound of the Formula I The compound ofthe Formula I forms cationic salts with pharmaceutically acceptablebases, such as, sodium hydroxide, potassium hydroxide, calcium hydroxidewhereby respectively sodium, potassium and calcium salts are obtained.Such addition salts, prepared by admixture ceutically acceptablecationic salts are useful as antibacterial agents and thus can combatgram-positive and gramnegative bacteria. They are also useful asantitrichomonal agents. They may be administered orally or parenterally,in conventional dosage forms with dosage adjusted to the individualneeds of the one treated.

The compound of the Formula I and its salts can be used in the form ofconventional pharmaceutical preparations. For example, said compoundscan be mixed with conventional organic and inorganic inertpharmaceutical carriers for parenteral or enteral administration suchas, for example, water, gelatin, starch, magnesium stearate, petroleumjelly or the like. They can be administered in conventionalpharmaceutical forms, for example, solid forms such as capsules,tablets, suppositories or liquid forms such as, solutions and emulsions.

The following examples are illustrative but not limitative of thepresent invention.

EXAMPLE 1 Cells of Pseudomonas aeruginosa ATCC-7700 were inoculated into6 liter Erlenmeyer flasks containing 2 liters of the inoculum mediumcomposed of (in g./liter); Baotopeptone. (The product resulting fromdigestion of proteinaceous material-meat casing or gelatin.) (Difco),6.0; N-Z amine type A (pancreatic hydrolysate of casein), 4.0; yeastautolyzate (National Yeast Products), 3.0; beef extract (Wilson), 1.5;and glucose, 1.0. The flasks were incubated at 28 for 72 hours on arotary shaker (240 r.p.m. with a 2-inch stroke). Four liters of inoculumwas then added to 1,320 liters of fermentation medium containing (ing./liter): Cerelose (crude glucose) (Corn Products), 11.0; Bacto-yeastextract (water soluble of autolyzed yeast) (Difco), 2,0; asparagine,0.5; and K HPO 0.5. The culture was incubated at 30 in a 1,900 literfermentor, aerated at 565 l.p.m. and agitated at 150 r.p.m. Siliconeantifoam (Dow Corning AF) was added as needed to control frothing. After42 hrs. the fermentation broth was clarified by Sharples centrifugation.

Isolation procedure The clarified broth was treated at pH 7.0 with 20g./ liter of charcoal (Darco G-60). After filtration through celite, thefiltrate was concentrated under reduced pressure to one-tenth itsoriginal volume. Five volumes of resin, chloride form). The resin wasconverted to the bicarbonate form by passing 6 liters of 3.7 M KHCOthrough the column. A charge of 25 g. of the so-obtained lyophilizedpowder dissolved in 200 ml. distilled water and adjusted to pH 10.0 with10% sodium hydroxide solution was then applied to the resin and theresin was eluted with 0.05 M trimethylammonim bicarbonate solution, pH9.5, prepared by sparging CO into 25% aqueous trimethylamine solutionfollowed by appropriate dilution. Fractions containing activity greaterthan 20 mcg./rnl., occurring at an elution volume of 4 liters to 9liters, were evaporated at reduced pressure to an oil. The oil was takenup in 7 ml. hot methanol and after storage for 48 hours at crystals ofL-Z-amino-4-methoxy-trans-3- butenoic acid were removed by filtration.After recrystallization from 80% aqueous ethanol, the product wasobtained, M.P. 240-245 (dec.); IR 1620 (carboxylate C=O), 1665 and 1220cm? (O-C=C); NMR (15 mg. ext. TMS), 6 7.25 (d., l., J=13 Hz., OCH=C),5.38 (d. of d., 1., 1:10 and 13 Hz., OH=Cg-C), 4.70 (d., 1., 1:10 Hz.,CH-CI NH 4.24 (s., 3, CE O); mass spectrum m/e 86 (only major peak); pKa2.47 pKa 9.55; [@1 3 +115 (c.=l, H O); 0rd (c.=0.13, 1 N []7oo=+ []5a9=+[]2sa=i+ (P []244= [l215= Analysis.Calcd for C H NO (percent): C, 45.71;H, 6.92; N, 10.68; OCH 23.66. Found (percent): C, 45.96; H, 6.94; 10.58;OCH 22.66.

EXAMPLE 2 A six-inch diameter column was filled to a height of 90 cm.with 25 liters Dowex 1X4 resin, (SO-100 mesh, chloride form) and 100gal. of 5% NaHCO solution was passed through the column to convert theresin to the bicarbonate form. After washing with 100 gal. of 0.1%aqueous ammonia solution and backwashing with distilled water, the resinwas charged with 500 g. of lyophilized powder as obtained in Example 1dissolved in 6 liters distilled water adjusted to pH=9.5 with 25%aqueous trimethylamine solution. The resin was then eluted with 0.05 Mtriethylammonium bicarbonate solution (prepared as above). Fractionscontaining greater than mcg./ml. activity were combined in early andlate pools of elution volumes, 100150 liters and 150-200 litersrespectively. The L-2-amino-4-methoxy-trans-3-butenoic acid in thelatter, about 30% pure on a non-volatile solids basis, could becrystallized from methanol in up to 60% yield but the mother liquorsfrom this and the crude concentrate from the early pool required furtherpurification on a small anion exchange column as described above.

EXAMPLE 3 Parental formulation Each 1 cc. ampul contains: Per cc.L-2-amino-4-methoxy-trans 3 butenoic acid mg 5.1 Benzyl alcohol cc 0.1Water for injection, U.S.P., q.s. ad cc 1 Procedure (for 10,000 cc.):

(1) In a clean glass or glass-lined vessel, 8,000 cc. of Water forinjection were heated to 90 C. It was then cooled to 50-60 C., andbenzyl alcohol was added and dissolved with stirring. The solution wasthen allowed to cool to room temperature.

(2) The 51.0 grams of L-2-amino-4-methoxy-trans-3- butenoic acid wereadded under an atmosphere of nitrogen and stirred until completelydissolved.

(3) Sufficient water for injection was then added to make a total volumeof 10,000 cc.

(4) This solution was then filtered through an 02 Selas candle, filledinto suitable size ampules, gassed with nitrogen and sealed.

4 EXAMPLE 4 5.0% cream Mg. per gramL-2-arnino-4-methoxy-trans-3-butenoic acid 50.00

Stearyl alcohol 100.00

Cetyl alcohol 15.00 White petrolatum 70.00 Methyl parahydroxybenzoate,U.S.P. 2.00 Propyl parahydroxybenzoate, U.S.P. 0.50 Isopropyl palmitate60.00 Polyoxyl 40 stearate, U.S.P 40.00 Propylene glycol 120.00 Disodiumversenate 0.10 Distilled water 548.16

Procedure:

(1) The stearyl alcohol, cetyl alcohol, petrolatum, propylparahydroxybenzoate, isopropyl palmitate and polyoxyl 40 stearate weremelted at 75 C. The mixture was cooled to and maintained at 70 C.

(2) Disodium versenate and methyl parahydroxybenzoate were dissolved inhot distilled water to which was added the propylene glycol. Thesolution was mixed at 75 C. and slowly added to the oil solutionprepared previously, using slow agitation. The emulsion was graduallycooled with slow stirring.

(3) When the temperature of the ointment reached 55 C., a solution ofL-2-amino-4-methoxy-trans-3-buten0ic acid was added and mixed with theointment.

(4) When the temperature of the ointment reached 50 C., cold water wascirculated in the jacket of the kettle and the ointment was cooled to 30C. with stirring. The ointment was then transferred to storagecontainers.

EXAMPLE 5 Table formulation Per tablet, mg.

L-2-amino-4-methoxy-trans-3-butenoic acid Lactose, U.S.P. 202 Cornstarch, U.S. P. 80 Amijel B0ll* 20 Calcium stearate 8 Total weight 401"A prehydrolyzed food grade corn starch. Any similar prehydrolyzed cornstarch may be used. Purchased from: Corn Products Company, 10 E. 56thSt., New York, NY.

Procedure:

(51) L-2-amino-4-methoxy-trans-3-butenoic acid, lactose, corn starch,and Amijel B011 were blended in a suitable mixer.

(2) The mixture was granulated to a heavy paste with water and the moistmass was passed through a #12 screen. It was then dried overnight at F.

(3) The dried granules were passed through a #16 screen and transferredto a suitable mixer. The calcium stearate was added and mixed untiluniform.

(4) The mixture was compressed at a tablet weight of 410 mg. usingtablet punches having a diameter of approximately (Tablets may be eitherflat or biconvex and may be scored if desired.)

EXAMPLE 6 Capsule formulation Per capsule, mg.

L-2-amino-4-methoxy-trans-3-butenoic acid 10 Lactose, U.S.P. Cornstarch, U.S.P. 30 Talc,U.S.P 5

Total weight 210 Procedure:

(11) L-2-amino-4-methoxy-trans-3-butenoic acid, lactose and corn starchwere mixed in a suitable mixer.

(2) The mixture was further blended by passing through 5 6 a Fitzpatrickcomminuting machine with a #1A screen References Cited with knivesforward.

(3) The blended powder was returned to the mixer, the Tobie: N. Bact. 52685 (1946). talc added and blended thoroughly- Shiro et al.: CA. 7344909q 1970 (4) The mixture was filled into #4 hard shell gelatin 5capsules on a Parke Davis capsulating machine. (Any LORRAINE A.WEINBERGER, Primary Examiner similar type capsulating machine may beused.)

What is claimed is: I. F. TEMPANE, Assistant Examiner 1. A compound ofthe formula 011 0 H 10 US. Cl. X.R.

19s -s0 R; 424-319 H C-NH2 H/ \C 0 0H and pharmaceutically acceptablecationic salts thereof. 15

